Certain 3-ketosteroids used to inhibit steroid 5α-reductase

ABSTRACT

The invention discloses 3-ketosteroids of the following formula used to inhibit steroid 5α-reductase: ##STR1## wherein R is OH, C 1  -C 6  alkanoyl, C 1  -C 6  alkanoyloxy, C 1  -C 4  alkanol, COCH 2  OH, CO 2  H, CONR 7  R 8 , cyclopropoxy, acetylthioalkane, cyclopylamino, 2-2-dimethyldioxolan-4-yl, 1,2-dihydroxyethyl and C 1-4  alkanethiol; 
     R 1  is hydrogen, hydroxy or C 1-6  alkyl; 
     R 1  and R 2  together may indicate ═O, that is an oxygen double bonded to the 17 carbon; 
     R 2 , R 3  and R 4  are each independently hydrogen or C 1-6  alkyl; 
     R 5  and R 6  are each independently hydrogen or OH; 
     R 5  and R 6  together may indicate ═O, that is an oxygen double bonded to the 11 carbon; 
     R 7  is hydrogen or C 1-8  alkyl; 
     R 8  is C 1-8  alkyl; and 
     with the proviso that, when R is OH, then R 1  is hydrogen; and with the proviso that, when R 5  is OH, then R 6  is hydrogen.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. Ser. No. 08/737,031 filedFeb. 11, 1997, now U.S. Pat. No. 5,750,744 which is a CIP of Ser. No.08/231,433 filed May 2, 1994, now abandoned, and also a U.S. Chapter 2filing of PCT/US95104399 which has an effective international filingdate of Apr. 11, 1995.

BACKGROUND OF THE INVENTION

The invention relates generally to androgenic and/or estrogenicinhibitor compounds, their use in the inhibition of C₁₇₋₂₀ lyase, C₁₇α-hydroxylase and 5α-reductase, and a method for the preparation of4-amino-Δ⁴ -3-ketosteroids.

Androgenic and estrogenic biosynthesis is principally controlled by theaction of the dual acting enzyme steroid C₁₇₋₂₀ lyase and C₁₇α-hydroxylase. While C₁₇₋₂₀ lyase catalyses the conversion of steroidshaving a two carbon side chain at the 17β-position, C₁₇α -hydroxylaseplaces a hydroxyl group of such a molecule at the 17α-position. Theaction of C₁₇₋₂₀ lyase creates important precursor molecules to theformation of testosterone, 5α-dihydrotestesterone and the estrogens,principally estrone and estradiol. Effective inhibition of C₁₇₋₂₀ lyasewould be useful in inhibiting the formation of both androgenic andestrogenic steroids, and thus is useful in the treatment of diseasestates or disorders where said androgens and/or estrogens play anadverse role.

The enzyme steroid 5α-reductase catalyzes the conversion of testosteroneinto dihydrotestosterone or DHT (17β-hydroxy-5α-androstan-3-one). DHT isa more potent androgen than testosterone and acts as an end-organeffector in certain tissues, particularly in mediating growth. Effectiveinhibition of this enzyme would be useful in preventing the formation ofDHT, which thus is useful in the treatment of androgen dependentdisorders, particularly those in which DHT plays a principal adverserole.

As the previously mentioned inhibitors affect various steps of theandrogenic and/or estrogenic pathway, each with known therapeuticutility in the treatment of various androgen and/or estrogen dependentdisorders, an alternative technique for the synthesis of said inhibitorswould also be useful. Certain of the 4-aminosteroid derivatives whichmay be obtained by the process described in this application aredisclosed in U.S. Pat. No. 4,757,061, issued Jul. 12, 1988, U.S. Pat.No. 5,120,840, issued Jun. 9, 1992 and U.S. Pat. No. 5,143,909, issuedSep. 1, 1992. The disclosed three step synthesis of these compoundsinvolves formation of a 4,5-epoxysteroid derivative followed bytreatment with sodium azide to provide the 4-azidosteroid derivative.The 4-azidosteroid derivative is subsequently reduced to provide the4-aminosteroid derivative. The use of sodium azide in this synthesisinvolves health risks due to the inherent instability of the compound. Askilled chemist can safely carry out the above process on a small scalein the laboratory, because only a small quantity of sodium azide isused. However, the large scale industrial production of the4-aminosteroid derivative requires large amounts of sodium azide and itsderivative acid, hydrazoic acid. This synthesis, which requires largequantities of sodium azide and hydrazoic acid at elevated temperaturesposes significant risks to human life and the environment. Theenvironmental and health risks could be reduced through appropriatedesign of a chemical plant, however the cost of such a facility would beprohibitive and the inherent risks could still not be entirelyeliminated. The three step synthesis of a 4-amino-A⁴ -steroid via the 4azido-intermediate is graphically illustrated in Scheme A. ##STR2##

Previous attempts at nitrating Δ⁴ -3-ketosteroids, described by Schaub,et al. in Tetrahedron 20:373 (1964) and by Suginome et al. J. Bull.Chem. Soc. Jap. 62:1343 (1989), resulted in formation of thecorresponding 2-nitrosteroid. This is suggested to have occurred becausethe nitration is effected through the kinetic 2,4-dienolate rather thanthe 3,5-dienolate which is the thermodynamic dienolate. The creation of4-nitrosteroids would require conditions which are supportive togeneration of a 3,5-dienolate as opposed to the 2,3-dienolate.

SUMMARY OF THE INVENTION

The present invention provides a novel process for the preparation of4-amino-3-ketosteroids via the formation of 4-nitro-3-ketosteroids whichare the product nitration of the corresponding 3,5-dienolate.

The present invention provides a novel process for preparing a compoundof the formula: ##STR3## wherein R is OH, C₁ -C₆ alkanoyl, C₁ -C₆alkanoyloxy, C₁ -C₄ alkanol, COCH₂ OH, CO₂ H, CONR₇ R₈, cyclopropyloxy,cyclopropylamino, acetylthioalkane, 2,2-dimethyldioxolan-4-yl,1,2-dihydroxyethyl, and C₁₋₄ alkanethiol;

R₁ is hydrogen, hydroxy or C₁₋₆ alkyl;

R and R₁ together may indicate ═O, that is an oxygen double bonded tothe 17 carbon;

R₂, R₃ and R₄ are each independently hydrogen or C₁ -C₆ alkyl;

R₅ and R₆ are each independently hydrogen or OH;

R₅ and R₆ together may indicate ═O, that is an oxygen double bonded tothe 11 carbon;

R₇ is hydrogen or C₁ -C₈ alkyl;

R₈ is C₁ -C₈ alkyl; and

the notation - - - on the ring indicates that the bond may be a singleor double bond;

the notation ##STR4## indicates a substituent in the α-configuration(below the plane of the paper);

the notation ##STR5## indicates a substituent in the β-configuration(above 35 the plane of the paper);

wherein further the above is limited:

with the proviso that when R is OH, then R₁ is hydrogen; and

with the proviso that, when R₅ is OH, then R₆ is hydrogen; comprisingsequentially:

a) reacting a starting compound of the formula ##STR6## wherein R-R₈and - - -, ##STR7## are defined as above, with an effective amount of astrong base at an elevated or suitable temperature for a time sufficientto generate the corresponding thermodynamic dienolate, followed byaddition of a neutral nitrating agent to produce a 4-nitrosteroid, andthen;

b) reacting the 4-nitrosteroid with a suitable reducing agent; and

c) optionally converting the 4-aminosteroid into a pharmaceuticallyacceptable salt.

Where post nitration modifications are required these can be made in theusual manner, by known procedures. For example, oxidation of the C₁₇hydroxyl group of 4-nitrotestosterone gives the corresponding 17-ketoneas depicted in the following scheme: ##STR8##

Removal of protecting groups can be effected as shown in the followingscheme: ##STR9##

The present invention further provides certain 4-nitrosteroids which areuseful as inhibitors of C₁₇₋₂₀ lyase, C₁₇α -hydroxylase and/or5α-reductase. These compounds are represented by the formula: ##STR10##wherein R is OH, C₁ -C₆ alkanoyl, C₁ -C₆ alkanoyloxy, C₁ -C₄ alkanol,COCH₂ OH, CO₂ H, CONR₇ R₈, cyclopropyloxy, cyclopropylamino,acetylthioalkane, 2,2-dimethyldioxolan-4-yl, 1,2-dihydroxyethyl and C₁₋₄alkanethiol;

R₁ is hydrogen, hydroxy or C₁₋₆ alkyl;

R and R₁ together may indicate ═O, that is an oxygen double bonded tothe 17 carbon;

R₂, R₃ and R₄ are each independently hydrogen or C₁ -C₆ alkyl;

R₅ and R₆ are each independently hydrogen or OH;

R₅ and R₆ together may indicate ═O, that is an oxygen double bonded tothe 11 carbon;

R₇ is hydrogen or C₁ -C₈ alkyl;

R₈ is C₁ -C₈ alkyl; and

the notation - - - on the ring indicates that the bond may be a singleor double bond;

the notation ##STR11## indicates a substituent in the α-configuration(below the plane of the paper);

the notation ##STR12## indicates a substituent in the β-configuration(above the plane of the paper);

wherein further the above is limited:

with the proviso that when R is OH, then R₁ is hydrogen; and

with the proviso that, when R₅ is OH, then R₆ is hydrogen.

The present invention further provides to a method of inhibiting steroidC₁₇₋₂₀ lyase, C₁₇α -hydroxylase and/or 5α-reductase which comprising theadministration of an effective inhibitory amount of the compounds of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The starting compounds are known or may be obtained by known techniques.

As used herein the term "C₁ -C₆ alkanoyl" refers to a straight orbranched chain alkanoyl radical of from one to six carbon atoms.Included within the scope of this term are formyl, acetyl, propionyl,butyryl, isobutyryl, hexanoyl and the like.

As used herein the term "C₁ -C₄ alkanol" refers to a straight orbranched chain alcohol radical of from one to four carbon atoms,containing at least one hydroxy functional group, but no more than 1hydroxy group attached to each carbon atom. Included within the scope ofthis term are methanol, ethanol, n-propanol, isopropanol, n-butanol,2-butanol, 2-methyl-l-propanol, 2-methyl-2-propanol,1,2-dihydroxyethanol, 1,3-dihydroxyisopropanol and the like.

As used herein the term "C₁ -C₆ alkyl" refers to a saturated straight orbranched chain hydrocarbon radical of from one to six carbon atoms.Included within the scope of this term are methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and thelike.

As used herein the term "C₁ -C₈ alkyl" refers to a saturated straight orbranched chain hydrocarbon radical of from one to eight carbon atoms.Included within the scope of this term are methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl,n-octyl and the like.

As used herein, the term "C₁₋₄ alkanethiol" refers to a saturatedstraight or branched chain hydrocarbon radical of from one to fourcarbon atoms containing a thiol functional group. Included within thescope of this term are methylthiol, ethylthiol, propylthiol,isopropylthiol, n-butylthiol, isobutylthiol, t-butylthiol and the like.

As used herein, the term "acetylthioalkane" refers to a saturatedstraight or branched chain hydrocarbon radical of from one to eightcarbon atoms containing an acetylthiol functional group. Includedwithing the scope of this term are acetylthiomethyl, acetylthioethyl,acetylthiopropyl, acetylthioisopropyl, acetylthio-butyl,acetylthio-s-butyl, acetylthio-t-butyl and the like.

As used herein, the term "C₁ -C₆ alkanoyloxy" refers to a saturatedstraight or branched chain hydrocarbon radical of from one to eightcarbon atoms containing a carboxylato functional group. Included withthe scope of this term are formyloxy, acetoxy, propionyloxy,isopionyloxy, n-butyryloxy, s-butyryloxy, t-butyryloxy, n-pentanoyloxy,s-pentanoyloxy, t-pentanoy, n-hexanoyloxy and the like.

As used herein, the term "pharmaceutically acceptable salts" is readilydeterminable by one of ordinary skill in the art and means an acidaddition salt which does not pose a significant toxic effect to thepatient and which possesses desirable pharmaceutical handling andformulation properties. Such salts can be either inorganic or organicand may be hydrated or substantially anhydrous. Illustrative inorganicacids which form suitable salts include hydrochloric, hydrobromic,sulphuric, phosphoric acid and metal salts such as sodium monohydrogenorthophsphate and potassium hydrogen sulfate. Illustrative organic acidswhich form suitable salts include the mono, di and tri carboxylic acids.Illustrative of but not limited to such acids are for example, acetic,glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic,tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic,hydroxybenzoic, phenylacetic, cinnamic, salicyclic, 2-phenoxybenzoic,and sulfonic acids such as methane sulfonic acid and 2-hydroxybenzoicand 2-hydroxyethane sulfonic acid. ##STR13##

The above reaction scheme illustrates the process of the invention,wherein a Δ⁴ -3-one steroid is nitrated to the corresponding 4-nitrocompound first by reaction with an effective amount of a basesufficiently strong to generate the Δ³,5 -dienolate. By "effectiveamount of a strong base," it is meant a base of strength andconcentration suitable to convert the Δ⁴ -3-one starting compound intoits thermodynamic dienolate under suitable reaction conditions.

In the selection of suitable reaction conditions, the exact base andconcentration thereof is a function of other reaction conditions, suchas the number and types of substituents, the reaction time, the solventand the temperature used. That said, general guidelines for theparameters of the formation of the thermodynamic dienolate intermediatevia deprotonation at the steroid carbon 6 are illustrated in thefollowing text. The range of usable "strong bases" include metalalcoholates and the like, but preferably are the salts of branched chainalcohols of 3 to 6 carbons. Most preferably, these branched chainalcohols contain 3 to 5 carbons. For example, potassium tert-butoxide.

The "effective amount" of strong base is variable depending upon thenumber of acidic protons on the steroid. An "acidic proton" is one whichreadily dissociates, for example, OH, CO₂ H, NH. For each acidic protonan additional equivalent of base is used. Using the typical basesdescribed herein and "non-acidic proton" containing steroids, the amountis at least two (2) molar equivalents relative to the steroidalcompound. Preferably it is two to four molar equivalents, and mostpreferably it is about two (2) molar equivalents, exclusive of acidicprotons.

In the creation of the dienolate intermediate in the nitration reaction,one of ordinary skill will recognize the sliding range betweentemperature and the time of the reaction. At elevated temperatures onewould expect shorter reaction times, while at lower temperatures, longerreaction times. In the practice of the invention, an "elevatedtemperature" is between about 50° C. to about 100° C., preferablybetween 50° C. and 83° C., and a most preferred reaction temperature isabout 83° C. At elevated temperatures, the "time sufficient to createthe corresponding thermodynamic dienolate" is between about 15 minutesand 8 hours, preferably between about 15 minutes and about 180 minutes,and a most preferred reaction time is about 60 minutes.

At lower temperatures a "suitable temperature" ranges from about 15° C.to about 50° C., preferably 17° C. to about 30° C. and most preferablyfrom about 20° to about 25° C. At these temperatures, the "timesufficient to create a thermodynamic dienolate" is between about 15minutes and 48 hours, preferably about 1 hour to 24 hours, and mostpreferred about 10 to 24 hours. Reaction yields can be increased byusing lower reaction temperatures with longer reaction times, and arepreferred in the practice of the invention. Since certain solvents whichare advantageously used with the process of the invention may have afreezing point at the lower end of above temperature range, it may benecessary to slightly warm and or mix the solvent with one of thereactants before the reaction can procede at these lower temperatures.

Solvents suitable to effect the nitration can be any alcohol derivedfrom straight or brached chain alkanes containing from two (2) to five(5) carbon atoms. Particularly desirable are secondary and tertiaryalcohols. For example, while ethanol or isopropanol may be usedeffectively, tert-amyl alcohol and tert-butyl alcohol are preferred. Themost preferred solvent is tert-butanol.

The nitration can be effected by any neutral nitrating agent, forexample, alkyl nitrates. The choice of a particular alkyl nitrate isdetermined by considerations of reactivity and cost. Suitable alkylnitrates are any saturated straight or branched chain organo-nitratecontaining from three to eight carbon atoms. Preferable alkyl nitratesinclude isopropyl nitrate, isobutyl nitrate and 2-ethyl hexylnitrate.The most preferred suitable alkyl nitrate is isopropyl nitrate.

The reduction of the 4-nitrosteroid compound by a suitable reducingagent may be effected by any known means, including, for example, eitherchemically or catalytically. Typical chemical catalysts include: 1) zincmetal in acetic acid; 2) zinc metal in methanol either in the presenceor absence of ammonium chloride; 3) stannous chloride in ethanol; or 4)iron and acetic acid in ethanol. An effective catalytic system includesLindlar's catalyst (palladium on calcium carbonate "poisoned" with leadand quinoline) in an alcohol solvent such as ethanol. Other systems maybe used in the catalytic hydrogenation, for example palladium oncharcoal and ammonium formate in methanol, palladium on charcoal andtrifluoroacetic acid in ethanol, or palladium on barium sulfate inethanol.

The invitro enzymatic inhibitory activity of the present compounds asinhibitors of steroid C₁₇₋₂₀ lyase was established using microsomalpreparations of the enzyme from rat or cynomolgus monkey testiculartissue. Microsomes were isolated from cynomolgus monkey or rattesticular tissue. The compound to be tested was dissolved in dimethylsulfoxide and diluted in 0.05M potassium phosphate buffer, (pH 7.4 forcynomolgus monkey lyase activity and pH 7.2 for rat lyase activity) togive the desired concentrations of test compound. The final assayconcentration of DMSO was 0.1% (v/v). Assays contained an NADPHregenerating system comprised of 1M NADPH, 5 mM glucose-6-phosphate, 1IU/mL glucose-6-phosphate dehydrogenase and microsomal protein in atotal volume of 0.2 mL.

For determination of time dependent C₁₇₋₂₀ lyase inactivation, the testcompound was incubated with 20 to 62 μg/mL microsomal protein, 0.05Mpotassium phosphate buffer, pH 7.4, and the NADPH regenerating systemdescribed above at 34° C. for 0 to 40 minutes. Aliquots of 180 μL werethen removed and assayed for enzyme activity. Each aliquot was added to7-³ H!-17α-hydroxypregnenolone (11.2 mCi/mmole; 0.2 μCi per assay) plusunlabeled 17α-hydroxypregnenolone to give a total substrateconcentration of 0.3 μM per assay and subsequently incubated for 6minutes at 34° C. For determination of reversible inhibition by the testcompound, the reaction was initiated by the addition of substrate andinhibitor (or DMSO in buffer for controls) simultaneously to the otherassay components. The substrate used for cynomolgus monkey lyase was 7-³H-17α-hydroxypregnenolone, which yielded a final concentration of 0.3 μMsubstrate. For assay of rat lyase activity, the substrate used was 1,2-³H!-17α-hydroxyprogesterone to give a total substrate concentration of0.1 μM (K_(m) =0.095 μM). The complete assay was incubated at 34° C. for6 minutes for both rat and monkey lyase.

The activity of the present compounds as inhibitors of steroid5α-reductase was determined using microsomal preparations of the5a-reductase enzyme from laboratory animal prostate tissue.Specifically, microsomes were isolated from cynomolgus monkey prostatetissue. Protein concentration of the microsomal preparations wasdetermined prior to use of the samples. Individual assays of cynomolgusmonkey prostatic 5α-reductase activity contained 0.1M potassiumphosphate-sodium citrate buffer, pH 5.6, 0.1% bovine serum albumin(w/v), 1.0 mM sodium EDTA, 7 to 96 mg of microsomal protein, 1.0 mMNADPH, 5.0 mM glucose-6-phosphate, 1 IU/mL glucose-6-phosphatedehydrogenase, 1,2-3H!-testosterone (0.15 μCi), unlabeled testosteroneto yield the desired concentration of substrate, and inhibitor which wasdissolved in DMSO then diluted in 0.1M potassium -sodium citrate buffer(50:50), pH 5.6, to yield a final assay concentration of 0.1% (v/v)DMSO. The same buffer and DMSO without inhibitor were used in controlassays. Background radioactivity was determined from assays containingall components except enzyme. Assays were performed in duplicate. Thereaction was initiated by the addition of testosterone and incubated for30 minutes at 25° C. in a Dubnoff® shaker incubator. The total volume ofthe assay was 100 μL. The assay was linear with time to 30 minutes underthese conditions.

Compound to be tested for inhibition was added simultaneously withtestosterone. For IC₅₀ determinations, a single concentration oftestosterone at the K_(m) level was used. The K_(m) values oftestosterone were determined in multiple experiments, and ranged from0.025 μM to 0.091 μM for cynomolgus 5α-reductase.

Each assay was terminated by addition of 5 mL of chloroform:methanol(2:1). Carrier steroid (2.5 μg each) representing substrates andproducts and 0.8 mL of distilled, deionized water were then added toeach assay. Carrier steroids for the lyase assays were17α-hydroxy-pregnenolone, dehydroepiandrosterone, andandrost-5-ene-3β,17β-diol (cynomolgus monkey lyase assays) or17α-hydroxyprogesterone, androstenedione, and testosterone (rat lyaseassays). Testosterone, dihydrotestosterone, and 3,17-androstanediol wereadded to the 5α-reductase assays as carrier steroids. Radiolabeled andunlabeled steroid were extracted by the method of Moore and Wilson(Methods in Enzymol., eds. O'Malley, B. W. and Hardlan, J. G. 36, 1975,pp. 466-473). The organic phase containing the steroids was evaporatedusing nitrogen gas. For lyase assays, the residues dissolved in 18%tetrahydrofuran (v/v) in hexane. For 5α-reductase assays, the driedsteroid residues were dissolved in 3% (v/v) isopropanol in hexane. Thesteroids were then separated by normal phase HPLC on a LiCrosorb® DIOLderivatized silica gel column (10 μm; 4×250 mm) with a 3% to 7.5%isopropanol in hexane gradient, followed by isocratic conditions of 75%(v/v) isopropanol in hexane. Radioactivity in the steroid peaks wasmeasured using a Radiomatic® Model HS or Model A515 Flo-One detector forboth lyase and 5α-reductase assays.

The enzyme activity for each assay was calculated from the percentconversion of substrate to products, and the results were expressed aspercent inhibition of control. The data from these experiments werefitted into the appropriate two parameter model incorporating sixconcentrations of inhibitor to determine an IC₅₀ value. When thecompounds were tested using the above procedures, the following resultswere obtained: (20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one exhibitedan IC₅₀ of 41 nM for monkey testicularC₁₇₋₂₀ lyase, 86 nM for rattesticular C₁₇₋₂₀ lyase and 17 nM prostatic monkey 5α-reductase. Whenthe compounds were tested using the above procedures with cynomolgusmonkey testicular lyase, the following results were obtained:

    ______________________________________                 Preincubation                            Concentration    Compound     Time       (μM)    % Inhibition    ______________________________________    17β-cyclopropyloxy-4-                 0          1          78    nitroandrost-4-en-3-one 0.1        36                 40         1          95                            0.1        79    (20S)-20-    0          1          69    mercaptomethyl-4-       0.1         0    nitropregn-4-en-3-one                 40         1          93                            0.1        26    ______________________________________

In the inhibition of steroid C₁₇₋₂₀ lyase, 5α-reductase and/or C₁₇α-hydroxylase the "effective inhibitory amount" is such amount ofcompound wherein an enzyme inhibitory effect is achieved. The exactamount necessary to achieve the desired inhibitory level is a functionof enzyme concentration, surface area, temperature and other typicalexperimental parameters, and is within experimental variation of one ofordinary skill in the art. Typically, when such compounds areadministered to actual patients, the minimal effective amount is suchamount where therapeutic effect is achieved. The exact amount ofcompound to be administered will vary over a wide range, dependingprincipally upon patient type and size. For example, depending on thepatient to be treated, and the severity of the condition being treated,the effective inhibitory amount of compound administered can vary fromabout 0.625 to 62.5 mg/kg of body weight per day and is preferably fromabout 0.5 to 30 mg/kg of body weight per day. Unit dosages for oraladministration may contain, for example, from 10 to 500 mg of a compoundof the invention. Alternatively, the present compounds can beadministered by parenteral routes or by implants.

The following examples are presented to illustrate the present inventionbut they should not be construed as limiting it in any way. As usedherein, unless described otherwise, all references to "room temperature"shall mean about 20° C. to about 23° C.

Step One: Preparation of 4-Nitrosteroids

EXAMPLE 1

(20S)-20-Hydroxymethyl-4-nitropregn-4-en-3-one

Potassium tert-butoxide (1.70 g, 15 mM) and(20S)-20-hydroxymethylpregn-4-en-3-one (1.65 g, 5 mM) are mixed togetherin tert-butanol (25 mL), and heated at reflux temperature under argonatmosphere for 75 minutes. Iso-propylnitrate (0.51 mL, 5 mM) is thenadded to the refluxing reaction mixture, resulting in an exothermicreaction. After one minute, the reaction vessel is removed from heat andallowed to cool to room temperature. The cooled mixture is then madeacidic by addition of acetic acid (5 mL), and stirred overnight. Themixture is then diluted with dichloromethane so as to form a separatephase sufficient to solubilize the synthesized product. Subsequently,the solids are removed by filtration and washed with dichloromethane.The materials in both the filtrate and wash(es) are combined togetherand concentrated. The residue is then redissolved in dichloromethane,then purified by flash chromatagraphy on silica gel to give(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one. The crystallization ofthis material from aqueous acetone gives white needles (m.p. 166°-168°C.).

IR 3435, 1696, 1623(m), 1633 cm⁻¹.

MS(CI) 376(100%, M+1), 358(30%, M+1-H₂ O).

¹ H-NMR (CDCl₃) δ 0.73(3H, s, C₁₈ -Me), 1.06(d, C₂₀ -Me), 1.29(s, C₁₉-Me), 3.38(1H, dd, 1/2•C₂₁ -CH₂), 3.64(1H, dd, 1/2•C₂₁ -CH₂).

Analysis. Calculated for C₂₂ H₃₃ NO₄ •(0.2)H₂ O: C, 69.70; H, 8.80; N,3.62; Found: C, 69.78; H, 9.13; N, 3.40. This compound has the followingstructure: ##STR14##

EXAMPLE 1A

(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one

Potassium tert-butoxide (1.70 g, 15 mM),(20S)-20-hydroxymethylpregn-4-en-3-one (1.65 g, 5 mM) are mixed intert-butanol and heated at reflux temperature, under argon atmospherefor 90 minutes. The combination is then cooled to room temperature andtreated with one continuous portion of isopropylnitrate (0.51 mL, 5 mM).After 18 hours, the contents of the reaction vessel are acidified with 5ml of acetic acid, and subsequently diluted with dichloromethane. Thesolids are then removed by filtration and further washed with additionaldichloromethane. The filtrate and wash residue are purified as describedin Example 1 to give a crystallized(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one.

EXAMPLE 1B

(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one

Potassium tert-butoxide (1.70 g, 15 mM) and(20S)-20-hydroxymethylpregn-4-en-3 (1.65 g, 5 mM) are combined intert-butanol (25 ml) and stirred at room temperature for 3 hours underan argon atmosphere. To the combination is then added, as one continuousportion, iso-propylnitrate (0.51 ml, 5 mM ) after which the combinationis stirred overnight. Acetic acid (5 ml) is then added. After 1 hour,the solids remaining in the reaction vessel are filtered off and washedwith dicloromethane. The combined filtrate and washing residue are thenpurified as described in Example 1 to give crystallized(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one.

EXAMPLE 1C

(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one

Potassium tert-butoxide (7.6 g, 67.8 mM),(20S)-20-hydroxymethylpregn-4-en-3-one (6.6 g, 20 mM) in tert-butanol isheated at reflux temperature for 60 minutes under an argon atmosphere.Isobutylnitrate (2.34 ml, 20 mM) is then added in one continuousportion. After 20 minutes of further refluxing, the reaction vessel iscooled to room temperature. Acetic acid (10 ml) is then added, and thereaction vessel stirred for 18 hours, after which the combination isdiluted with dichloromethane (200 mL). The solids are removed, washed,and recombined with the washing residue and subsequently purified asdescribed in Example 1 to give crystallized(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one.

EXAMPLE 1D

(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one

Potasium tert-butoxide (7.6 g, 67.8 mM) and(20S)-20-hydroxymethylpregn-4-en-3-one (6.6 g, 20 mM) are combined intert-butanol and heated at reflux temperature for 120 minutes.2-ethylhexylnitrate (3.56 ml, 20 mM) is then added in one continuousportion. After 10 minutes, the reaction vessel is cooled to roomtemperature. The reaction is then quenched with acetic acid (9 ml) andstirred overnight. After diluting with dichloromethane (200 mL), thesolids are removed, washed, and combined with the washing residue andsubsequently purified as described in Example 1 to give crystallized(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one.

EXAMPLE 1E

(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one

React (20S)-Hydroxymethyl-pregn-4-en-3-one (6.18 g, 18.7 mmol) intert-butanol (91 mL) with potassium tert-butoxide (6.43 g, 57.3 mmol, 3molar equiv.) at reflux temperature for 1.5 hours. Add isopropyl nitrate(2.9 mL, 1.6 molar equiv.) dropwise over 20 minutes after which thereaction is allowed to cool to room temperature then stirred overnight(12-18 hours). The reaction mixture is then treated with acetic acid(6.2 mL), stirred for 1 hour at room temperature and poured into water(400 mL). Extract with dichloromethane (4×100 mL), combining all theorganic phases which is dried over magnesium sulfate. After filtering,evaporate the filtrate in vacuo providing a reddish oil, which ispurified by silica gel plug filtration (hexanes:Et₂ O:CH₂ Cl₂ =4:2:1,2.5 L then 2:2:1, 1 L). Combine and concentrate the appropriatefractions and recrystallize the resulting material from boilingisopropanol to which water is added until turbid to give the titlecompound (2.66 g).

EXAMPLE 2

17β-Hydroxy-4-nitroandrost-4-en-3-one

By the method of example 1, testosterone (2.88 g, 10 mM), potassiumtert-butoxide (3.40 g, 30 mM) and iso-propylnitrate (1.01 ml) arereacted to yield 17β-hydroxy-4-nitroandrost-4-en-3-one, m.p. 158°-160°C. (acetone-hexane)

IR 3533, 1689, 1615(m), 1635 cm⁻¹.

MS(CI) 334(100%, M+1), 316(50%, M+1-H₂ O).

¹ H-NMR δ (CDCl₃) 0.80(3H, s, C₁₈ -Me), 1.30(s, C₁₉ -Me), 3.66(1H, t,C₁₇ •H). This compound has the following structure: ##STR15##

The combined filtrate and washes from the above crystallization can betreated with acetic anhydride (3 mL) and pyridine (6 mL). After standingovernight at room temperature, the reaction is stirred with water for 1hour. A sticky solid is obtained by decantation of the liquids and thenpurified by chromatography to give17β-acetoxy-4-nitroandrost-4-en-3-one, m.p. 216°-217° C. (aqueousacetone).

IR (CHCl₃) 1725, 1695, 1623(m), 1536, 1256 cm⁻¹.

MS(CI) 376(100%, M+1), 316(35%, M+1-AcOH).

¹ H-NMR (CDCl₃) 0.84(3H, s, C₁₈ -Me), 1.30(s, C₁₉ -Me), 2.05(s, COMe),5.62(1H, dd, C₁₇ -H).

This compound has the following structure: ##STR16##

As suggested previously, the 17β-hydroxy-4-nitrosteroid may be modifiedinto the corresponding 17-one, such as described in the followingexample.

EXAMPLE 2A

4-nitroandrost-4-ene-3,17-dione

A solution of 17β-hydroxy-4-nitroandrost-4-en-3-one(4-nitrotestosterone, 10.56 g, 31.7 mM) in acetone (900 ml) cooled to-6° C. is treated with Jones reagent (10.0 ml). After the excess reagentis decomposed with methanol, the solids are removed by filtration. Thefiltrate is concentrated, for example on a rotary evaporator undervaccum, then purified by flash chromatography on silica gel to give4-nitroandrost-4-ene-3,17-dione, m.p. 205°-205.5° C.dec.(acetone-hexane).

IR 1738, 1622, 1533, 1373 cm⁻¹.

MS(CI) 332(100%, M+1).

¹ H-NMR(CDCl₃) 0.93(s, C₁₈ -Me), 1.13(s, C₁₉ -Me).

The compound has the following structure: ##STR17##

EXAMPLE 3

17β-hydroxy-7α-methyl-4-nitroandrost-4-en-3-one

17β-hydroxy-7α-methyltestosterone (11.2 g, 37.0 mM), potassiumtert-butoxide (8.29 g, 73.9 mM) and iso-propylnitrate (3.76 ml) arereacted by the method of example 1 to give17β-hydroxy-7α-methyl-4-nitroandrost-4-en-3-one, m.p. 229°-230° C.dec.(acetone-hexane).

IR (CHCl₃) 3614, 1694, 1658 (m), 1623, 1536 cm⁻¹.

MS(CI) 348 (100%, M+1), 330 (40%, M+1-H₂ O)

¹ H-NMR (CDCl₃) 0.89 (d, C₇ -Me), 0.91 (s, C₁₈ -Me), 1.16 (s, C₁₉ -Me),3.68 (t, C₁₇ -H).

The compound has the following structure: ##STR18##

The 17β-hydroxysteroid may be converted into the corresponding 17-one bythe procedure described in the following example.

EXAMPLE 3A

7α-methyl-4-nitroandrost-4-ene-3,17-dione

A solution of 17β-hydroxy-7α-methyl-4-nitroandrost-4-en-3-one (4.2 g,12.1 mM) in acetone (400 ml) is cooled to 0° C. and is treated withJones reagent (4.0 ml). Any excess reagent is decomposed by addition ofmethanol. The solids are filtered off and the filtrate is concentratedto a green solid. This material is purified by flash chromatography onsilica gel to give 7α-methyl-4-nitroandrost-4-ene-3,17-dione (aqueousacetone).

IR (CHCl₃) 1735, 1697, 1625(m), 1537 cm⁻¹.

MS(CI) 346(100%, M+1), 328(50%, M+1-H₂ O).

¹ H-NMR(CDCl₃) 0.86(3H, d, C₇ -H), 0.93(3H, S, C₁₈ -Me), 1.33(s, C₁₉-Me).

The compound has the following structure: ##STR19##

EXAMPLE 4

11α,17β-dihydroxy-17α-methyl-4-nitroandrost-4-en-3-one

11α,17β-dihydroxy-17α-methyl androst-4-en-3-one (6.37 g, 20.0 mM),potassium tert-butoxide (6.73 g., 60.0 mM) and iso-propyl nitrate (2.02ml) are reacted by the method of Example 1 to yield11α,17β-dihydroxy-17α-methyl-4-nitroandrost-4-en-3-one as a solid foam.

IR 3435, 1691, 1618(m), 1533, 1373 cm⁻¹.

MS(CI) 364(100%, M+1), 346(30%, M+1-H₂ 0), 328(37%, M+1-2H₂ O).

¹ H-NMR (CDCl₃) 0.93(3H, s, C₁₈ -Me), 1.21(s, C₁₉ -Me), 1.43 (s, C₁₇-Me), 4.08 (1H, dt, C₁₁ -H).

The compound has the following structure: ##STR20##

EXAMPLE 5

20,21-dihydroxy-4-nitropregn-4-en-3-one Acetonide

20,21-dihydroxypregn-4-en-3-one-21-acetate (6.73 g, 18.0 mM), potassiumtert-butoxide (5.78 g., 51.5 mM) and isopropyl nitrate (1.60 ml) arereacted by the method of Example 1 to give20,21-dihydroxy-4-nitropregn-4-en-3-one. Recrystallization from aqueousacetone yields the corresponding acetonide, m.p. 221°-223° C. (dec).

IR 1693, 1622(m), 1535, 1371 m⁻¹.

MS(CI) 418(55%, M+1), 101(100%).

¹ H-NMR (CDCl₃) 0.90(3H, s, C₁₈ -Me), 1.03(s, C₁₉ -Me), 1.33 (s, Me),1.37(s, Me), 3.46-3.54(1H, m, C₂₀ -H), 3.93-4.05 (2H, m, C₂₁ -CH₂).

The compound has the following structure: ##STR21##

The acetonide group can be removed from the 20,21 dioxygenated carbonsby the procedure of the following example.

EXAMPLE 5A

20,21-dihydroxy-4-nitropregn-4-en-3-one

A solution of 20,21-dihydroxy-4-nitropregn-4-en-3-one acetonide obtainedfrom the recrystallization filtrate of Example 5 in methanol is brieflywarmed to assist in forming a solution. The reaction vessel is thencooled to room temperature whereupon 5% aqueous hydrochloric acid (10mL) is added. After 4 hours of stirring, the reaction is neutralizedwith aqueous potassium carbonate and concentrated in vacuo. The residueis partitioned between dilute aqueous hydrochloric acid anddichloromethane. The organic layer is then separated, dried overmagnesium sulfate and concentrated to a yellow foam which is thenpurified by flash chromatography on silica gel to give20,21-dihyroxy-4-nitro-pregn-4-en-3-one, m.p. 183°-185° C. (aqueousmethanol).

IR (CHCl₃) 3628, 3587, 1693, 1622, 1535, 1373 cm⁻¹.

MS(CI) 378(100%, M+1), 342(35%, M+1-2H₂ O).

¹ H-NMR 0.92(3H, s, C₁₈ -Me), 1.30(3H, s, C₁₉ -Me), 3.34-3.44 (1H, m,C₂₀ -H), 3.61-3.72(2H, m, C₂₁ -CH₂).

The compound has the following structure: ##STR22##

EXAMPLE 6

17β-cyclopropyloxy-4-nitroandrost-4-en-3-one

17β-cyclopropyloxy-androst-4-en-3-one (9.70 g, 29.5 mM), potassiumtert-butoxide (7.0 g, 62.4 mM), and isopropyl nitrate (2.99 ml) arereacted by the method of Example 1 to yield17β-cyclopropyloxy-4-nitroandrost-4-en-3-one, m.p. 137°-38° C.(methanol).

IR (CHCl₃), 1695, 1622(m), 1535, 1373 cm⁻¹.

MS(CI) 374(100%, M+10, 316(20%, M+1-c-C₃ H₅ -O)

¹ H-NMR (CDCl₃) 0.38-0.61(4H, m), 0.80(3H, s, C₁₈ -Me), 1.29(s, C₁₉-Me), 3.25-3.33(1H, m, cyclopropyl-CHO), 3.44(1H, t, C₁₇ -H).

The compound has the following structure: ##STR23##

The starting material for the above nitration may be prepared asfollows:

A solution of 17β-cyclopropyloxy-androst-5-en-3β-ol (19.36 g, 58.9 mM)in acetone (1.9 L) is cooled to -3° C. and then treated with Jonesreagent (20 ml). The excess reagent is decomposed with methanol. Thesolids are removed by filtration. The filtrate is concentrated to agreen oil which is purified by flash chromatography on silica gel togive 17β-cyclopropyloxy-androst-4-en-3-one (11.0 g, 57%).

The starting material (17β-cyclopropyloxy-androst-5-en-3β-ol) for theabove oxidation may be prepared as described in U.S. Pat. No. 4,966,897to Angelastro and Blohm.

EXAMPLE 6A

17β-(cyclopropoxy)-4-nitro-androst-4-en-3-one

Potassium tert-butoxide (109 g, 0.97 mol, 2.1 molar equivalents) isadded to a stirred solution of 17β-(cyclopropyloxy)-androst-4-en-3-one(150 g, 0.46 mol) in tert-butanol (2 L) over 10 minutes at roomtemperature and under nitrogen. Continue stirring at room temperaturefor 18 hours, then add isopropylnitrate (48.2 g, 0.46 mol, 1.0 mol.equiv.) in tert-butanol (50 mL) over 30 minutes at room temperature.After an additional day of continuous stirring at room temperature,glacial acetic acid (130 mL) is added over 25 minutes and stirring iscontinued for an additional 18 hours. Subsequently, methylene chloride(1.5 L) and brine (saturated NaCl, 800 mL) is added and the solution isstirred an additional 10 minutes. The organic phase is then seperatedand dried over magnesium sulfate. The resulting slurry is filered andthe filtrate concentrated (35° C./40 Torr) to give a dark red oil.Purify by flash chromatography (SiO₂, elution: EtOAc/hexane 5:95,EtOAc/hexane 1:9 and EtOAc/hexane 15:85). Combine and concentrate thefractions containing the desired product (30° C./40 Torr) to give asolid residue which is stirred under hexane (350 mL). Filter and dry thesolids to give the title compound (68 g, 40%) as a yellow solid.Additional compound is obtained from the filtrate after concentrationand rechromatography as above (8 g, 5%). mp 133°-134° C.

IR (KBr) 3437, 3090, 2945, 2870, 1693, 1624, 1531, 1450, 1373, 1346,1332, 1211, 1188, 1170, 1076, 1062, 1035, 1012, 962, 794, 765 cm⁻¹.

1H-NMR (CDCl₃) δ 0.50 (4H, m, 2× cyclopropyl-CH2), 0.80(3H, s, C18-Me),1.30(3H, s, C19-Me), 3.3(3H, m, OCH of cyclopropyl), 3.44(1H, t, C17-H).

MS (CI, CH4) m/z (rel. intensity) 374 (100%, M+1).

Analysis calculated for C22H31NO4: C, 70.75; H, 8.37; N, 3.75; Found: C,70.99; H, 8.44; N, 3.56.

EXAMPLE 7

4-nitro-androst-4-en-3-one-17β-carboxylic acid

Androst-4-en-3-one-17β-carboxylic acid (3.63 g, 11.4 mmol), potassiumtert-butoxide (4g, 35.3 mmol ) and iso-propylnitrate (1.9 mL) arereacted by the method of Example 1 to yield4-nitro-21-androst-4-en-3-one-17β-carboxylic acid, m.p. 205°-208° C.dec.

IR (CHCl₃) 3034, 2970, 1697, 1535, 1373 cm⁻¹.

MS(CI) 362(100%, M+1).

¹ H-NMR δ (CDCl₃) 0.79(3H, s, C₁₈ Me), 1.3(3H, s, C₁₉ •Me).

The compound has the following structure: ##STR24##

EXAMPLE 8

3-hydroxy-4-nitroandrost-3,5-diene-17β-tert-butyl carboxamide

To a 250 mL round bottom flask equipped with a magnetic stirring bar anda gas inlet was placed potassium tert-butoxide (8.5 g, 75 mmole) andtert-butyl alcohol (75 mL). Androst-4-ene-3-one-17β-carboxamide (9.3 g,25 mmol) was added and the solution was stirred at 65° C. under an argonatmosphere for 20 minutes. Isopropyl nitrate (2.8 mL, 28 mmol) was addedcausing a strong exotherm and the solution was cooled to 25° C. over a 1hour period. Acetic acid (7.5 mL) was added, the precipitate wasfiltered, and the filtrate was concentrated. The resulting dark gum wasdissolved in dichloromethane, washed with water, concentrated, andchromatographed on 300 mL silica gel using 30-50% ethyl acetate/hexaneto give 441 mg (1.05 mmol) of crystalline product fromdichloromethane/hexane. m.p. 207°-208.5° C. (dec.);

IR (KBr): 3441, 2967, 2943, 2916, 2885, 2847, 1693, 1670, 1624, 1535,1508, 1475, 1452, 1390, 1367 cm⁻¹.

UV (EtOH) λ_(max) =242 nM; ε=13,100;

¹ H-NMR (CDCl₃): 64.40 (s, 1H), 2.52-2.60 (m, 2H), 1.4-2.46 (m, 16H),1.35 (s, 9H), 1.31 (s, 3H), 1.03-1.32 (m, 4H), 0.74 (s, 3H) ppm;

Analysis calc'd for C₂₄ H₃₆ N₂ O₄ : C: 69.20%; H: 8.71%, N: 6.72%; FoundC: 69.33%, H: 8.63%, N:6.60%.

The compound has the following structure: ##STR25##

EXAMPLE 9A

20-acetylthiomethyl-4-nitropregn-4-en-3-one

A solution of 20-hydroxymethyl-4-nitropregn-4-en-3-one (750 mg, 2.0mmol)(prepared in Example 1) and tosyl chloride (400 mg, 2.1 mmol) wasprepared in pyridine (2 ml) and stirred at 25° C. for 12 hours. After 24hours, the residue was redissolved in dichloromethane (2 ml) andadditional tosyl chloride (40 mg) added. After stirring another 12hours, 2 drops H₂ O were added and the mixture was stirred for 30minutes and washed with dichloromethane (50 ml), water (50 ml), 10% HCl(50 ml) and dried over MgSO₄. This material was concentrated andcrystallized from a 50:50 solution of ethyl acetate:hexane (1:1) to givea yellow solid. m.p. 181° C.-182° C.

The tosylate (529 mg) prepared by the above procedure was dissolved indry dimethylformamide (10 ml) and added to CsSCOCH₃ newly prepared bydissolving Cs₂ CO₃ (163 mg, 0.5 mmol) and HSCOCH₃ (86 mg, 1.1 mmol) inCH₃ OH (3 mL) and evaporating. After 24 hours, the reaction was dilutedwith diethyl ether, washed with water, concentrated and flashchromatographed using dichloromethane/hexane (4:1). The fractionscontaining the desired product were concentrated and crystallized fromhexane to give 225 mg thioester.

Alternatively, the tosylate prepared by the above procedure wasdisplaced to make the corresponding thioacetyl compound by the followingprocedure: Cs₂ CO₃ (163 mg, 0.5 mmol) was dissolved in methanol (2 mL)and HSCOCH₃ (90 mg, 1.1 mmol). The homogenous solution was concentratedin vacuo. The remaining residue was dissolved in dimethyl formamide (3mL) with the tosylate (529 mg, 1.0 mmol). The resulting solution wasstirred at 25° C. under N₂ for 18 hours. Another solution (1.0 mL ofdimethylformamide) of cesium thioacetate (1.0 mmol) prepared in theabove manner was added to the reaction vessel. Upon completion of theconversion (determined by TLC), 1N HCl (1 mL) was added and theresulting solution was dissolved in ethyl acetate, washed in water anddried over magnesium sulfate.

The compound has the following structure: ##STR26##

EXAMPLE 9B

20-mercaptomethyl-4-nitropregn-4-en-3-one

The thioacetate prepared in Example 9A (65 mg, 0.15 mmol) was dissolvedin 2 mL methanol and 1 mL THF with 0.3 mL of 1N LiOH. After stirring for1 hour, 0.1 mL of acetic acid was added. The solution was extracted intoethyl acetate, washed with water and dried over magnesium sulfate. Theproduct was dissolved into 1.0 ml acetic acid, heated to about 50° C.and concentrated in vacuo to give a light yellow solid (30 mg).

The compound had the following structure: ##STR27##

EXAMPLE 10

17β-cyclopropylamino-4-nitroandrost-4-en-3-one

A solution of 17β-cyclopropylamino-4-en-3-one (4.61 g, 14.07 mmol) andpotassium tert-butoxide (4.74 g, 42.22 mmol, 3 molar equivalents) intert-butanol (60 mL) was heated at reflux for 1 hour. Isopropyl nitrate(1.43 mL, 14.07 mmol, 1 molar equivalent) was added all at once as thesolution was refluxing. The reaction was slowly cooled to roomtemperature, after which glacial acetic acid (20 mL) and dichloromethane(20 mL) was added to the reaction mixture to dissolve the red-orangeprecipitate. The reaction was allowed to stand at room temperatureovernight. The reaction mixture was filtered and the filter cake washedwith dichloromethane until white. The filtrate was diluted withadditional dichloromethane (200 mL) and subsequently washed with anaqueous sodium chloride solution at one-half the saturated concentration(200 mL) followed by a washing of a solution consisting of equal partsaqueous half saturated sodium chloride and aqueous saturated sodiumbicarbonate (200 mL) . The organic layer was dried over magnesiumsulfate and concentrated in vacuo and purified by chromatagraphy onsilica gel (dichloro-methane/methanol, 19:1) to give17β-cyclopropylamino-4-nitroandrost-4-en-3-one as a solid yellow foam.

¹ H-NMR (300 MHz, CDCl₃) δ 0.75(s, 3H, C₁₉ -Me); 1.30 (s, 3H, C₁₈ -Me)ppm.

IR(KBr) 3435, 2944, 2870, 1695, 1533, 1371, 1013, 766 cm⁻¹.

MS(EI)=372 (M+).

The compound has the following structure: ##STR28## Step Two: Reductionof Steroidal 4-Nitro-Δ⁴ -3-ones into 4-amino-Δ⁴ -3-one SteroidalCompounds

A. Catalytic Reduction

EXAMPLE 11

4-amino-20-hydroxymethylpregn-4-en-3-one

A solution of 20-hydroxymethyl-4-nitropregn-4-en-3-one (2.01 g, 5.35 mM)in absolute ethanol (28 mL) is treated sequentially with Lindlar'scatalyst (5% Pd on CaCO₃ with 5.2% Pb, 0.81 g) and with quinoline (37μl) and kept under hydrogen at between 40-55 p.s.i. for 24 hours. Themixture is then filtered through celite, and the filtrate isconcentrated to a yellow solid which is purified by short pathchromatography to give 4-amino-20-hydroxymethylpregn-4-en-3-one, m.p.180°-185° C. (aqueous isopropanol).

IR 3512, 3470, 3384, 1648, 1614, 1576 cm⁻¹.

MS(CI) 346(100%, M+1), 328(30%, M+1-H₂ O).

¹ H-NMR 0.72(3H, s, C₈ -Me), 1.02(d, C₂₁ -Me), 1.15(s, C₁₉ -Me), 2.6-3.2(v.br, NH₂), 3.36(1H, dd, 0.5•C₂₂ -CH₂), 3.63(1H, dd, 0.5C₂₂ -CH₂). Thecompound has the following structure: ##STR29##

EXAMPLE 12

17β-cyclopropyloxy-4-amino-androst-4-en-3-one

A solution of 17β-cyclopropyloxy-4-nitroandrost-4-en-3-one (4.36 g, 11.6mM) in absolute ethanol (125 mL) was treated with Lindlar's catalyst andthen quinoline (80 mL). The mixture was stirred under hydrogen at 1atmosphere pressure for about 117 hours. The reaction mixture wasfiltered through celite topped with charcoal and washed with absoluteethanol. The combined filtrate and wash was concentrated to a brownliquid (3.7 g) and dissolved in methylene chloride, placed atop a columnof silica gel prepared with hexane/ethyl acetate (1:4) and purified byflash chromatography and eluting in hexane:ethyl acetate (1:4).

The product containing fractions were combined and concentrated to yielda light yellow glass which crystallized on standing (2.3 g). Thecrystals were dissolved in methanol, filtered through cotton, and waterwas added dropwise until crystallization began. The mixture wasrefrigerated overnight (12-18 hours). The crystals were collected byfiltration and washed with cold aqueous methanol and with water, thendried in vacuo to give 4-amino-17β-(cyclopropyloxy)-androst-4-en-3-oneas a light yellow solid (1.97 g).

IR(KBr) η3458, 3372, 1674, 1622 (m), 1585 cm⁻¹.

Anal. calc'd for C₂₂ H₃₃ NO₂ : C:76.92; H:9.68; N:4.08. Found: C:76.86;H:10.04; N:4.08.

¹ H-NMR (CDCl₃) δ 0.38-0.61 (4H, m, 2× CH₂), 0.79 (3H, s, C₁₈ -Me), 1.15(s,C₁₉ -Me), 3.27+3.44+ca.3.4 (4H, cycloproxy-H, C₁₇ -H, NH₂, m+t,v.br.).

UV(EtOH) λ 294(ε 7570, 1 g.ε 3.879).

MS/CI 344(100%, M+1), 286(30%, M+1-C₃ H₅ OH). The compound has thefollowing structure: ##STR30##

EXAMPLE 12A

4-amino-17β-(cyclopropoxy)-androst-4-en-3-one hydrochloride

To a stirred solution of 17β-(cyclopropyloxy)-4-nitro-androst-4-en-3-one(10 g, 26.7 mmol) in methanol (200 mL) at 35° C. was added Lindlar'scatalyst (4 g, 5.9% Pd+5.4% Pb/CCP3 (CaCO₃), D. R. Engelhard, Seneca,S.C.) and quinoline (0.2 g, 1.6 mmol) under a nitrogen atmosphere. Theresulting mixture was placed in a Parr shaker and shaken under ahydrogen atmosphere at room temperature under 50 psi for 20 hours,whereupon about 3 molar equivalents of hydrogen were consumed. Thecatalyst was removed by filtration.

The above procedure was repeated about 9 times (8×10 g-scale and 1×7-gscale), and the product solutions after filtration were combined.Silicon dioxide (SiO₂, 550 g) was added to the solution and the mixturewas concentrated under reduced pressure (10 Torr) and temperature(10°-15° C.). The mixture of SiO₂ and product was loaded onto a flashcolumn (20 cm i.d., containing 5 kg of SiO₂) and the column was elutedsequentially with 15% (20 L), 20% (20 L) and 30% (40 L) of ethyl acetatein hexane. The product containing fractions were pooled and concentratedat reduced temperature (10° C. and pressure (30 Torr) to give a yellowsolution (600 mL). The solution was treated with 1.8M HCl in ethylacetate (75 mL) at 4° C. The resulting slurry was diluted with acetone(200 mL) and CH₂ Cl₂ (100 mL). After stirring for 30 minutes, the solidswere collected by filtration and washed with acetone (300 mL) and driedto give the title compound (34 g, 37%); mp 206°-207° C. The filtrate wasconcentrated to a solution (300 mL) and the solid was collected to givea second crop of compound (3.4 g, 4%); mp 203°-204 °C.

IR (KBr) 3445, 3086, 2945, 2872, 2555, 1967, 1791, 1682, 1641 cm⁻¹.

¹ H-NMR (CDCl₃) δ 0.50(4H, m, 2×cyclopropyl-CH₂), 0.76(3H, s, C₁₈ -Me),1.21(3H, S, C₁₉ -CH₃), 3.2 (1H, m, OCH of cyclopropyl), 3.39 (1H, t, C₁₇-H), 9.6 (3H, br.s, NH₃).

MS (CI) m/z 344 (100%, M⁺).

Anal. calc'd for C₂₂ H₃₄ NO₂ Cl•(0.6) H₂ O: C, 68.05; H, 9.07; N, 3.61.Found C, 68.18; H, 9.06; N, 3.52.

EXAMPLE 13

17β-cyclopropylamino-4-aminopregn-4-en-3-one

17β-cyclopropylamino-4-nitropregn-4-en-3-one (670 mg, 1.80 mmol) wasdissolved in absolute ethanol (11 mL) and treated with Lindlar catalyst(268 mg) followed by quinoline (3 μL). The solution was stirredvigorously under a hydrogen atmosphere at atmospheric pressure (about760 mm/mg) for 18 hours. The reaction mixture was filtered and washedwith ethanol (100 mL) and dichloromethane (100 mL). The solvents wereremoved in vacuo and the product purified by chromatagraph on silica gel(CH₂ Cl₂ /CH₂ OH, 47:3) to give a yellow oil which crystallized to ayellowish solid. m.p. 149°-150° C. (Et₂ O).

IR(KBr): 3474, 3366, 2945, 1616, 1577 cm⁻¹.

MS(Cl/CH4) M⁺ +H!=343.

¹ H-NMR (33 MHz, CDCl₃) δ0.73(3H, s, C₁₈ -Me), 1.15(3H, s, C₁₉ -Me),2.66 (1H, t, C₁₇ -H).

¹³ C-NMR (75 MHz, CDCl₃) δ 6.464, 7.185, 11.289, 20.803, 23.682, 24.757,29.683, 29.753, 30.914, 32.860, 34.899, 35.307, 37.904, 42.453, 52.910,54.549, 69.014, 132.938, 138.860, 194.343.

The compound has the following structure: ##STR31##

B. Chemical Reduction

EXAMPLE 14

4-amino-20-hydroxymethylpregn-4-en-3-one

A solution of 20-hydroxymethyl-4-nitropregn-4-en-3-one (0.52 g, 1.38 ml)in absolute ethanol (4.8 mL) is combined with stannous chloride (2.1 g)added in one portion and then heated to 70° C. for 6 hours. The reactionvessel is then cooled to room temperature and the solution is carefullyneutralized with sodium bicarbonate (9 g) over a 10 minute period. Theresulting slurry is then filtered, removing a brown solid which is thenstirred in 10% hydrofluoric acid (25 mL) and ethyl acetate (25 mL). TheHF/EtOAc treatment is repeated. The organic phases from each filtrationare combined, dried over magnesium sulphate, filtered and concentrated.The resulting residue is purified by flash chromatagraphy to give4-amino-20-hydroxymethylpregn-4-en-3-one as a white solid identical tothe material described in U.S. Pat. No. 5,218,110 to Weintraub and U.S.Pat. No. 5,120,840 to Weintraub et al., which are both hereinincorporated by reference.

The compound has the following formula: ##STR32##

EXAMPLE 14

4-amino-17-cyclopropyloxyandrost-4-en-3-one

A solution of 17-cyclopropyloxy-4-nitroandrost-4-en-3-one (1.0 g, 2.71mM) in acetic acid (10 mL) is treated with zinc dust (1.0 g). Thecombination is vigorously stirred for 1.5 hours at room temperature. Thezinc salts are removed by filtration and washed with ethyl acetate. Thecombined filtrate and wash are combined and concentrated to a yellowsolid which is then redissolved in ethyl acetate and extracted threetimes with 1M hydrochloric acid (150 ml). The combined acid extracts areneutralized with sodium hydroxide (pH 14) and further extracted withether. The combined organic layers are then dried over sodium sulfateand concentrated to give 4-amino-17-cyclopropyloxyandrost-4-en-3-one(0.59 g), m.p. 100°-102° C. (aqueous methanol).

IR 3354, 1662, 1620, 1581 cm⁻¹.

MS(CI) 344(100%, M+1).

¹ H-NMR 0.37-0.61(4H, m, 2×cyclopropyl CH₂), 0.79(3H, s, C₁₈ -Me),1.16(s, C₁₉ -Me), 3.25-3.33(m, cyclopropyl-CHO), 3.44(t, C₁₇ -H).

The compound has the following structure: ##STR33##

EXAMPLE 16

20-acetylthiomethyl-4-aminopregn-4-en-3-one

20-(Thioacetyl)methyl-4-nitropregn-4-en-3-one (173 mg, 0.40 mmol) and300 mg zinc dust were stirred in 2 mL glacial acetic acid for 30minutes. The mixture was poured into ethyl acetate (50 mL), washed with3×50 mL of a saturated aqueous solution of sodium bicarbonate and driedover magnesium sulfate. m.p. 173° C.-176° C.

The compound had the following structure: ##STR34##

What is claimed is:
 1. A method for inhibiting steroid 5α-reductasewhich comprises administering to a patient in need thereof an effectiveinhibitory amount of a compound of the formula:wherein R is OH, C₁ -C₆alkanoyl, C₁ -C₆ alkanoyloxy, C₁ -C₄ alkanol, COCH₂ OH, CO₂ H, CONR₇ R₈,cyclopropoxy, acetylthioalkane, cyclopylamino,2-2-dimethyldioxolan-4-yl, 1,2-dihydroxyethyl and C₁₋₄ alkanethiol; R₁is hydrogen, hydroxy or C₁₋₆ alkyl; R₁ and R₂ together may indicate ═O,that is an oxygen double bonded to the 17 carbon; R₂, R₃ and R₄ are eachindependently hydrogen or C₁₋₆ alkyl; R₅ and R₆ are each independentlyhydrogen or OH; R₅ and R₆ together may indicate ═O, that is an oxygendouble bonded to the 11 carbon; R₇ is hydrogen or C₁₋₈ alkyl; R₈ is C₁₋₈alkyl; andwith the proviso that, when R is OH, then R₁ is hydrogen; andwith the proviso that, when R₅ is OH, then R₆ is hydrogen.
 2. A methodaccording to claim 1 wherein the compound to be administered is(20S)-20-hydroxymethyl-4-nitropregn-4-en-3-one.